Method for identifying biologically active oligonucleotides capable of modulating the immune system

ABSTRACT

The present invention relates to methods of identifying oligonucleotides capable of modulating the immune system in a mammalian subject, comprising analysis of which tertiary structural type said oligonucleotide adopts, in phosphate-buffered saline solution. Further, the invention provides oligonucleotides identifiable by the methods of the invention and to their use in methods of treating diseases, such as inflammatory diseases, autoimmune diseases, infectious diseases, neurodegenerative diseases and cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.13/995,304 filed on Jun. 18, 2013, which is the National Phase of PCTInternational Application No. PCT/EP2011/073468 filed on Dec. 20, 2011and claims priority under 35 U.S.C. §119(a) to patent application Ser.No. 10/196,290.0 filed in Europe on Dec. 21, 2010, patent applicationSer. No. 10/196,273.6 filed in Europe on Dec. 21, 2010, and patentapplication Ser. No. 10/196,285.0 filed in Europe on Dec. 21, 2010, allof which are hereby expressly incorporated by reference into the presentapplication.

FIELD OF THE INVENTION

The present invention refers to methods of identifying oligonucleotidesthat adopts a certain tertiary structure, and thereby are able tomodulate the immune system, irrespective of primary structure. Thepresent invention relates to methods of identifying oligonucleotidescapable of modulating the immune system in a mammalian subject,comprising analysis of which tertiary structural type saidoligonucleotide adopts, in physiologically relevant aqueous solution.Further, the invention provides oligonucleotides identifiable by themethods of the invention and to their use in methods of treatingdiseases, such as inflammatory diseases, autoimmune diseases, infectiousdiseases, neurodegenerative diseases and cancer.

BACKGROUND

DNA oligonucleotides (oligodeoxyribonucleotides, oligodeoxyribonucleicacids, ODNs, oligonucleotides) are short DNA-based synthetic polymersthat can be synthesised and highly purified in significant quantities.The sequence of monomers (deoxyribonucleotides) in oligonucleotides istermed as the primary structure of DNA. The secondary structure of anucleic acid molecule refers to the base-pairing interactions within asingle molecule or set of interacting molecules. The tertiary structureof DNA is determined as its spatial organization (IUPAC). ODNs inphysiologically relevant aqueous solutions are considered to berandom-coiled single-stranded or in the tertiary structure ofdouble-stranded DNA helix.

The double helix is the dominant tertiary structure for biological DNAthat can be in one of three DNA conformations and are believed to befound in nature, A-DNA, B-DNA, and Z-DNA. The B-form described by Watsonand Crick is believed to predominate in cells (Richmond T. J., et al.(2003) Nature 423 (6936): 145-150). However, several types of nucleicacid structures can be observed that are different from random orclassical double-stranded helix forms. Among them are triplexes,quadruplexes and other nucleic acid structures (Soyfer, V. N and PotamanV. N. (1995) Triple-Helical Nucleic Acids. Springer Ver., New York, 360pp; Burge S., et al. (2006) Nucleic Acids Research, 34, 19, 5402-5415).

Recently, it has been found that particular G-rich DNA sequences arecapable of forming stable four-stranded structures known asG-quadruplexes (G-quartets) (Burge S., et al. (2006) Nucleic AcidsResearch, 34, 19, 5402-5415; Huppert, J. L. (2008) 37(7):1375-84; Neidleand Balasubramanian (2006) Quadruplex Nucleic Acids, RSC Publishing,Cambridge, UK, 302 pp). G-quartets arise from the association of fouradjacent G-bases assembled into a cyclic conformation. These structuresare stabilized by von Hoogsteen hydrogen bonding and by base stackinginteractions (Skogen M., et al., (2006) BMC Neuroscience 7:65).G-quadruplexes have been shown to be relevant in biological processes asbeing important components of human telomeres, and playing a role in theregulation of transcription as well as translation (Patel et al., (2007)Nucleic Acids Res. 35(22):7429-55; Oganesian L, and Bryan T M (2007)Bioessays 29(2):155-65; Qin and Hurley (2008) Biochimie., 90(8):1149-71; Siddiqui-Jain et al., (2002) Proc Natl Acad Sci USA. (2002)3; 99(18):11593-8; Kumari et al., (2007) Nat Chem Biol. 2007,3(4):218-21).

Parekh-Olmedo et al., ((2004) J Mol Neurosci. 24(2):257-67) showed thatcertain groups of ODNs can inhibit pathological protein aggregation inHuntington's disease. One of these groups was the G-richoligonucleotides (GROs). G-quartet formation has also been implicated inthe non-antisense antiproliferative effects of GROs. In several cases,the biological effects of oligonucleotides designed as antisense agentswere found to be unrelated to inhibition of target protein expression,but instead were associated with the formation of G-quartet structures(Burgess et al., (1995) Proc. Natl. Acad. Sci. USA, 92, 4051-4055;Anselmet, A., et al., (2002) FEBS Lett., 510, 175-180; Benimetskaya, L.,et al., (1997) Nucleic Acids Res., 25, 2648-2656; Saijo, Y et al.,(1997) Jpn J. Cancer Res., 88, 26-33).

The molecular mechanisms of GRO action are not fully known. One of themappears to be related to the ability of oligonucleotides to bind tonucleolin (Bates, P. J., et al. (1999) J. Biol. Chem., 274,26369-26377). Binding of nucleolin to other G-quartet-forming sequencessuch as telomeric DNA, immunoglobulin switch regions and ribosomal geneshas also been reported (Dempsey, L. A., et al., (1999) J. Biol. Chem.,274, 1066-1071; Hanakahi, L. A. et al., (1999) J. Biol. Chem., 274,15908-15912; Ishikawa, F. et al., (1999) Mol. Cell. Biol. 13, 4301-4310;Dickinson, L. A. and Kohwi-Shigematsu, T. (1995) Mol. Cell. Biol., 15,456-465).

Treatment of tumour cells with G-rich oligonucleotides was found toinhibit cell cycle progression by specifically interfering with DNAreplication, whereas GRO-treated normal skin cells exhibited minimalperturbation of the cell cycle (Xu X., et al., (2001) J. Biol. Chem.276, 43221-43230). Further, Antisoma plc, developed G-quadruplex basedAS-1411 that is the first oligodeoxyribonucleotide aptamer that reachedclinical trials for the potential treatment of cancers, including acutemyelogenous leukemia (AML) (Ireson CR and Kelland L R, (2006) Mol CancerTher. 5 (12):2957-62; Mongelard F. and Bouvet P., (2006) Curr Opin MolTher. 12(1): 107-14).

G-rich oligonucleotides can form a variety of possible quadruplexstructures, depending on its thermodynamic and kinetic characteristics.Quadruplexes can be formed by one, two or four molecules ofoligonucleotides, which are referred to as monomer, dimer and tetramerstructures, respectively. (Dapic V., et al., (2003) Nucleic AcidsResearch 31 (8): 2097-2107).

Circular dichroism (CD) spectroscopy is commonly used to investigate thestructure and conformation of nucleic acids (Baase and Johnson Jr.(1979) Nucleic Acids Res., 6(2): 797-814; Giraldo R. et al., (1994)Proc. Natl. Acad. Sci. USA 91: 7658-7662; Hardin C. C. et al., (1991)Biochemistry 30:4460-44721992, Hardin C. C. et al., (1992) Biochemistry31: 833-841; Paramasivan S, et al. (2007) Methods 43: 324-331) wherecircular dichroism refers to the differential absorption of left andright circularly polarized light (P. Atkins and J. de Paula (2005)Elements of Physical Chemistry, 4th ed. Oxford University Press).Various DNA quadruplex structures have distinctive circular dichroismspectra (Dapic V, et al., (2003) Nucleic Acids Research 31 (8):2097-2107) providing the possibility to use selected structures as setof standards or references to compare with CD spectra ofoligonucleotides.

Various immunostimulatory oligodeoxyribonucleotides containingunmethylated deoxyribodinucleotide CpG motifs (CpG ODNs) that mimicprokaryotic DNA have been developed and characterised by severalresearch groups. It has been established that recognition of CpG ODNrequires Toll-like receptor 9 (TLR9) interaction. Cells that expressTLR9, which include plasmacytoid dendritic cells (PDCs) and B cells,produce Th1-like proinflammatory cytokines, interferons, and chemokinesin response to CpG ODNs. Several classes of CpG ODNs are described up todate as A-, B-, C-, D- and P-class CpG ODNs (Krieg A., 2002 and 2006),however, they all have been classified based on the primary structure(nucleotide sequence) of the oligonucleotides.

In recent years, there has been tremendous progress delineating thespecific components of the immune system that contribute to variousaspects of normal immunity and specific disease states. This hasintroduced the possibility to treat diseases with immunomodulatingsubstances as protein therapeutics, including monoclonal antibodies andcytokines, which became mainstream treatments in a number of clinicalsettings.

Imbalances in the cytokine cascade can help the initiation andpropagation of the immune driven inflammation. In several inflammatorydiseases, including rheumatoid arthritis and inflammatory bowel disease,the proinflammatory cytokine TNF-α has been shown to play a central rolein inflammatory reactions and has proven to be an especially attractivetarget for biological agents. Immunomodulatory cytokines considered ofsignificance in the treatment of infectious diseases, malignancies andautoimmune diseases including interferon type I (IFN-α and IFN-β), IFN-γand IL-10.

Interferons (IFNs) are cytokines that may be released in response toviruses, bacteria, parasites and tumor cells. Interferons possessimmunoregulatory, antiviral and anti-cancer properties. They have beenused to successfully treat a number of chronic inflammatory disordersincluding multiple sclerosis (Paolicelli, D et al., (2009) Targets &Therapy; 3, 369-76), chronic viral hepatitis (Hoofnagel J H and Seeff LB, (2006) N. Eng. J. Med., 355: 2444-51; Chevaliez S and Pawlotsky J M,(2009) Handbook of Experimental Pharmacology, Antiviral Strategies, 189:203-41) and also in neoplastic diseases (Gill P S et al., (1995) N. Eng.J. Med., 332:1744-8). There are two main classes of IFNs: Type I IFNs(α,β,ε,o,κ) are central in the host defense against pathogens such asviruses whereas type II IFN (γ) mainly contributes to theT-cell-mediated regulation of the immune responses.

IFN-α is produced by the cells of the immune system in response to thepresence of a foreign antigen, inducing cell activation of macrophagesand natural killer cells and enhancing antigen presentation. There are13 subtypes of IFN-α, whereby the two subtypes IFN-α2a and IFN-α2b havebeen used therapeutically with similar results in hepatitis C (Welzel TM et al., (2009) Hepatology, 49: 1847-58) and renal carcinoma (Coppin Cet al., (2008) The Cochrane Collaboration, Targeted therapy for advancedrenal cell carcinoma, 1-38). The side effects of recombinant IFN-α can,however, be significant with up to 68% of patients presenting withpsychiatric symptoms, such as depression, irritability, and insomnia.

IFN-β is produced mainly in fibroblasts and plasmacytoid dendritic cellsand has 30% nucleic acid homology to IFN-α and sharing similar antiviralactivity. Clinically, it has been used in the treatment of MS because ofits additional anti-inflammatory effect (Durelli L et al., (2009) AnnNeurol, 65: 499-509). Currently, recombinant IFN-β is used as afirst-line treatment for relapsing-remitting form of the MS disease.Common adverse events from the recombinant IFN-β are depression,flu-like symptoms, and increase of liver enzyme levels. In addition,treatment results in the induction of anti-IFN-β neutralizing antibodies(NAbs) in some patients resulting in a lost effect of treatment(Soelberg Sorensen Petal., (2003) Lancet, Vol. 362: 1184-91; SoelbergSorensen P et. al., (2006) Neurology, 67: 1681-3). IFN-β was also usedsuccessfully as therapy in chronic inflammatory diseases as ulcerativecolitis (Musch E et al., (2002) Aliment Pharmacol Ther, 3: 581-6).

IFN-γ is produced by leukocytes to induce macrophage activation andincrease oxidative burst. Defects in IFN-γ and IFN-γ receptor genes havebeen associated with autoimmune diseases such as rheumatoid arthritis,type1 diabetes and multiple sclerosis (Chen J and Liu X, (2009) CellularImmunology, Vol. 254: 85-90). However, treatment of autoimmune diseasessupplementing with IFN-γ was ambivalent due to its broad biologicaleffects causing unwanted activities. Further, it is clinically used toenhance immunity in patients with chronic granulomatous disease withgood efficacy. Potential side effects include fever, hypotension, andflu-like symptoms (Holland S M, (2009) Clinic Rev Allerg Immunol, 38:3-10). It is also thought to be beneficial as treatment for brain tumorimmunotherapy (Hague A et al., (2007) Neurochem Res, 32: 2203-2209).

Interleukins are a group of multifunctional cytokines that are producedby a variety of lymphoid and non-lymphoid cells of the immune system tomediate communication between the immune cells and are particularlyimportant to promote immune responses as inflammation and in thehematopoeisis. An example of a proinflammatory classified interleukin isIL-6. Its dysregulation can contribute to the induction and maintenanceof several diseases such as rheumatoid arthritis and inflammatory boweldisease (Heinrich P C et al., (2003) Biochem. J., 1374: 1-20). IL-6 hasalso anti-inflammatory properties by for example inhibiting TNFs (Opal SM and DePalo V A, (2000) Chest Anti-inflammatory cytokines, 117: 932-4)reflecting the challenge of using a cytokine as therapy or as target forimmunotherapy. In contrast, IL-10 is classified as an anti-inflammatorycytokine and is produced by monocytes, macrophages, mast cells, T and Blymphocytes, and dendritic cells. It is believed that it can suppressthe production of pro-inflammatory cytokines and plays a central role inthe regulation of immune responses. It also has broad implications inthe development of certain inflammatory diseases, most noticeablyallergy and asthma (Hawrylowicz C M and O'Garra A, (2005) Nat RevImmunol, 202: 1459-63; Ogawa Y et. al., (2008) Curr Mol Med, 8: 437-45).Numerous clinical studies have indicated that there is a general lack ofsufficient levels of IL-10 in asthmatic patients which may contribute toa more intensive inflammation as shown by K. Tomita and colleagues whodescribed that levels of IL-10 and IL-10 producing cells weresignificantly reduced in patients with severe persistent asthma whencompared to mild asthma (Tomita K et al., (2002) Clin Immunol, 102:258-66). It is also believed that corticosteroids, widely usedanti-inflammatory compounds, exert their anti-inflammatory effects inpart by enhancing IL-10 production (Richards D F et al., (2000) Eur JImmunol, 30: 2344-54). In corticosteroid resistant asthmatic patients,corticosteroids failed to induce IL-10 synthesis suggesting a stronglink between induction of IL-10 synthesis and efficacy ofcorticosteroids (Hawrylowicz C M et al., (2002) J Allergy Clin Immunol,109: 369-70). Experiments from D. Hesse and colleagues (Hesse D et al.,(2010) Europ. J. Neurol., 15: 1-7) indicated that the expression ofendogenous IFN-β induces the expression of IL-10 in MS and that theexpression of IL-10 negatively correlates with the disease activitysuggesting that IL-10 expression is associated with the dampening of theinflammatory response. Furthermore, in patients with evolvedneutralizing antibodies (NAbs), IL-10 expression is reduced.

The use of cell surface antigens as therapeutic targets is anothergrowing area of modulating the immune system. Using antibody-relatedtherapies can have several options such as binding to a specific targetmolecule on the cell surface to trigger cellular mechanisms such asapoptosis or activation pathways (immunotherapy), or simply binding to atarget on the cell surface for delivery of an agent to the specific celltype, e.g. cytostatic agent (immuno-chemotherapy). Immunotherapy is usedin the treatment or alleviation of many immunological diseases orconditions, such as cancer, inflammatory diseases such as asthma andallergy and also autoimmune disorders such as multiple sclerosis.

WO2010/053433 A1 describes the potential of specific oligonucleotides inup-regulating the expression of certain cell surface markers or cellsurface antigens such as CD20, CD23, CD69 and CD80. The pre-incubationof PBMC isolated from CLL patients significantly increased the rate ofapoptosis in human B-cells mediated by a monoclonal antibody directedagainst CD20 (rituximab).

WO2010/053430 A1 describes the capability of specific oligonucleotidesto influence the properties and behaviour of polymorphonuclear cells, inparticular the recruitment and/or migration of polymorphonuclear cellsto a site of inflammation, and that they through this mechanism haveutility in the prevention, treatment and/or alleviation of variousdiseases such as ischemia.

The challenges of immunotherapy and treatment with cytokines are theoccurring side effects and the observed immunogenicity of these proteintherapeutics even of fully human protein drugs (Vial T and Descotes J,(1994) Drug Saf, 10: 115-20; Scott D W and De Groot A S, (2010) ANNRheum Dis, 69: 72-76). Especially, for the treatment within IFN-α it wassuggested that the efficacy of the treatment has to be increased whilethe toxicity should be decreased (Sarasin-Filipowicz M, (2010) Swiss MedWkly, 140: 3-11). The usage of endogenous induced IFN-α could be moreeffective and tolerable. P. Sfriso and colleagues could for example showthat exposure to fungi is positive for the treatment of inflammatorybowel disease (Sfriso Petal., (2010) J Leuk Biol, 87: 385-95). Fungi area natural source of foreign DNA and proteins, inducing endogenouscytokine production. Endogenous induction of cytokines could givebeneficial effects without unwanted induced activities. Another aspectis that IFN-α, for example, exists in numerous subforms, however throughendogenously induced expression all subforms will be expressed in theirnatural way. It could be shown in corticosteroid-resistant asthmapatients that IL-10 is up-regulated after IFN-α treatment and theauthors suggest that the beneficial effects of IFN-α lies in theproduction of IL-10 (Simon H U et al., (2003) Allergy, 58: 1250-1255).IL-10 has less side effects than IFN-α and therefore could be a bettertreatment option. Chen and colleagues described the opposite functionsof IL-10 and IFN-γ in a subform of CD4+ T-cells while they are workingtogether in the disease management of chronic infections (Chen J and LiuX S, (2009) J Leuk Biol, 86: 1305-10). These examples demonstrate howdifferent cytokines with different biological functions can act togetherto modulate the pathogenesis of a disease or to maintain the finebalance in an immune response. A more effective treatment option couldbe a combination of different cytokines or a way to induce differentcytokines endogenously. There is clearly a need to provide methods andoligonucleotides that can induce the endogenous expression of specificcytokines.

DESCRIPTION OF THE INVENTION

Although synthesized oligonucleotides may interact with the cellularreceptor (TLR9), the inventors have surprisingly identified that varioussynthetic oligonucleotides can induce different patterns of cytokineexpression in human PBMCs wherein the functionality is dependent on thetertiary structure of the oligonucleotides and not on the primarystructure or the content of any specific sequence feature as for examplethe dinucleotide CpG. Non-CpG oligonucleotides can induce cytokineexpression and some CpG containing oligonucleotides cannot. Theinventors have developed a method for identifying new oligonucleotidesthat can induce preferably specific cytokines, wherein saidfunctionality is dependent on their tertiary structure. Theoligonucleotides would be useful in treatment of diseases related todeficiencies or imbalance in these cytokines.

The present invention refers to methods of identifying oligonucleotidesthat adopts a certain tertiary structure, and thereby are able tomodulate the immune system, irrespective of primary structure. Theprimary structure of the oligonucleotides identifiable by the methods ofthe invention may or may not fall into any of previously known ODNprimary structure classes but as long as they have the desired tertiarystructure described by the invention, they are unified by both thetertiary structure and their capability to modulate the immune system,such as inducing particular cytokines.

In the present invention, there is provided methods for identifyingoligonucleotides capable of modulating the immune system in a mammalianscomprising analyzing which tertiary structural type said oligonucleotideadopts, in phosphate-buffered saline solution. Typically, the method iscapable of identifying oligonucleotides characterized in that they format least 40% of telomeric G-quadruplex tetramer type of tertiarystructure. This group of oligonucleotides are surprisingly active instimulation of IFNs compared to similar oligonucleotides (in primarystructure) that do not form a telomeric G-quadruplex tetramer type oftertiary structure. Such oligonucleotides with nearly identical primarystructure surprisingly differ strongly in their capability of inducingIFNs production. Particularly the ODNs identifiable by the methods ofthe present invention form at least 40% of telomeric G-quadruplextetramer type of tertiary structure, independently of their primarystructure and these are able to induce specific cytokine profiles, inparticular IFN-α, IFN-β and/or IFN-γ. Further, the method is capable ofidentifying oligonucleotides characterized in that they form at least45% of non-G-quadruplex dimer type of tertiary structure. This group ofoligonucleotides are surprisingly active in stimulation of cytokinesIL-6 and/or IL10, compared to similar oligonucleotides (in primarystructure) that do not form a non-G-quadruplex dimer type of tertiarystructure. Such oligonucleotides with nearly identical primary structuresurprisingly differ strongly in their capability of inducing IL-6 and/orIL10 production. Particularly the ODNs identifiable by the methods ofthe present invention form at least 45% of non-G-quadruplex dimer typeof tertiary structure, independently of their primary structure andthese are able to induce specific cytokine profiles, in particular IL-6and/or IL-10.

The inventors have set out to develop a method for identification ofnovel oligonucleotides capable of endogenously modulating the immunesystem in a mammalian subject depending on the tertiary structureadopted in solution and, therefore useful in the treatment ofinflammatory and autoimmune diseases. Other objects underlying theinvention, as well as advantages associated with the invention, willbecome evident to the skilled person upon study of the description,examples and claims.

SHORT DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H depict the aligned CD spectra of reference oligonucleotides(refs #1-8) used in the calculation of the relative structuralcomposition of the CD spectra of the oligonucleotides of the invention.The wavelength of the spectra is represented on the abscissa axis andcorresponding molar ellipticity value is represented on the ordinateaxis.

FIGS. 2A-2G depict the molar ellipticity of 3 oligonucleotides withmajor contribution telomeric G-quadruplex tetramer type of tertiarystructure (IDX-9054, IDX-9059, IDX-9133), 3 oligonucleotides with lessthan 40% contribution of telomeric G-quadruplex tetramer type oftertiary structure (IDX-0445, IDX-0465, IDX-9134) and a negative controlsample (IDX-9011). The experimental data overlaid on the calculated CDspectra input of standard components; CD spectra indicated as follows:sample—experimental measured spectra of oligonucleotide, #1—standardcomponent 1 (SEQ ID NO 1); #2—standard component 2 (SEQ ID NO 2);#3—standard component 3 (SEQ ID NO 3); #4—standard component 4 (SEQ IDNO 4); #5—standard component 5 (SEQ ID NO 5); #6—standard component 6(SEQ ID NO 6); #7—standard component 7 (SEQ ID NO 7); #8—standardcomponent 8 (SEQ ID NO 8). The samples represent oligonucleotides withSEQ ID NO 7 (IDX-9011); SEQ ID NO 81 (IDX-9054); SEQ ID NO 91(IDX-0445); SEQ ID NO 59 (IDX-0465); SEQ ID NO 83 (IDX-9059); SEQ ID NO80 (IDX-9133); and SEQ ID NO 94 (IDX-9134). The wavelength of thespectra is represented on the abscissa axis and corresponding molarellipticity value is represented on the ordinate axis.

FIGS. 3A-3J depict the molar ellipticity of 8 oligonucleotides withmajor contribution of non-G-quadruplex dimer type of tertiary structure(IDX-0910, IDX-0912, IDX-9022, IDX-0475, IDX-0480, IDX-9071, IDX-0001,IDX-9024), one oligonucleotide with less than 45% contribution ofnon-G-quadruplex dimer type of tertiary structure (IDX-0465) and anegative control sample (IDX-9011). The experimental data overlaid onthe calculated CD spectra input of standard components; CD indicated asfollows: sample—experimental measured spectra of oligonucleotide,#1—standard component 1 (SEQ ID NO 1); #2—standard component 2 (SEQ IDNO 2); #3—standard component 3 (SEQ ID NO 3); #4—standard component 4(SEQ ID NO 4); #5—standard component 5 (SEQ ID NO 5); #6—standardcomponent 6 (SEQ ID NO 6); #7—standard component 7 (SEQ ID NO 7);#8—standard component 8 (SEQ ID NO 8). The samples representoligonucleotides with SEQ ID NO 7 (IDX-9011); SEQ ID NO 56 (IDX-0910);SEQ ID NO 57 (IDX-0912); SEQ ID NO 44 (IDX-9022); SEQ ID NO 10(IDX-0475); SEQ ID NO 11 (IDX-0480); SEQ ID NO 47 (IDX-9071); SEQ ID NO48 (IDX-0001); SEQ ID NO 49 (IDX-9024) and SEQ ID NO 59 (IDX-0465). Thewavelength of the spectra is on the abscissa axis and correspondingmolar ellipticity value is on the ordinate axis.

FIGS. 4A-4B represent an example of calculation of the relativecomposition of the CD spectra of IDX 9022 (SEQ ID NO 44) applying afitting analysis using computer program for the mathematicaldecomposition of experimental data. FIG. 4A: Molar ellipticity of IDX9022 (SEQ ID NO 44) overlaid on the aligned experimental measured CDspectra of the oligonucleotides used as standard components in thecalculation of the relative structural composition of the CD spectra ofthe samples. FIG. 4B: Molar ellipticity of IDX 9022 (SEQ ID NO 44)overlaid on the aligned theoretically calculated fitting CD spectra ofstandard components and on the fitting curve of sum of the theoreticallycalculated CD spectra of standard components. Inserted table indicatescalculated fitting CD spectra input (%) of standard components. CDspectra indicated as follows: sample—experimental measured spectra ofIDX 9022 (SEQ ID NO 44) oligonucleotide, fit—fitting curve of sum of thecalculated CD spectra input of standard components, #1—standardcomponent 1 (SEQ ID NO 1); #2—standard component 2 (SEQ ID NO 2);#3—standard component 3 (SEQ ID NO 3); #4—standard component 4 (SEQ IDNO 4); #5—standard component 5 (SEQ ID NO 5); #6—standard component 6(SEQ ID NO 6); #7—standard component 7 (SEQ ID NO 7); #8—standardcomponent 8 (SEQ ID NO 8). The wavelength of the spectra is on theabscissa axis and corresponding molar ellipticity value is on theordinate axis.

FIGS. 5A-5B represent an example of calculation of the relativecomposition of the CD spectra of IDX 9054 (SEQ ID NO 81) applying afitting analysis using computer program for the mathematicaldecomposition of experimental data. FIG. 5A: Molar ellipticity of IDX9054 (SEQ ID NO 81) overlaid on the aligned experimental measured CDspectra of the oligonucleotides used as standard components in thecalculation of the relative structural composition of the CD spectra ofthe samples. FIG. 5B: Molar ellipticity of IDX 9054 (SEQ ID NO 81)overlaid on the aligned theoretically calculated fitting CD spectra ofstandard components and on the fitting curve of sum of the theoreticallycalculated CD spectra of standard components. Inserted table indicatescalculated fitting CD spectra input (%) of standard components. Thewavelength of the spectra is on the abscissa axis and correspondingmolar ellipticity value is on the ordinate axis.

FIG. 6A represents the molar ellipticity of IDX 9057 (SEQ ID NO 77) withmajor contribution (64.0%) of telomeric G-quadruplex tetramer type oftertiary structure, which oligonucleotide is linked dominantly with IFNinduction. FIG. 6B represents the molar ellipticity of IDX 9147 (SEQ IDNO 33) with major contribution (97.0%) of non-G-quadruplex dimer type oftertiary structure, which oligonucleotide is linked dominantly withIL-6/IL-10 induction.

DETAILED DESCRIPTION OF THE INVENTION

Before the invention is described in detail, it is to be understood thatthis invention is not limited to the particular compounds described orprocess steps of the methods described since compounds and methods mayvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It must be noted that, as used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a sequence” includes morethan one such sequence, and the like.

Further, the term “about” is used to indicate a deviation of +/−2percent of the given value, preferably +/−5 percent and most preferably+/−10 percent of the numeric values, when applicable.

The phrase “capable of modulating the immune system” is used to describea function, wherein a substance is capable to alter an immune response.

Immunomodulation is an alteration, adjustment or regulation of theimmune response. An immunomodulator is a substance that has an effect onthe immune system in form of immunostimulation or immunosuppression orboth. The modulation of the immune system may represent an increase or abalancing of the levels of cytokines. This effect can be mediated by,but is not limited to, cytokines (lymphokines, chemokines, interleukins,interferons), cell surface markers, receptors, prostaglandins andhormones. These changes in the immune response can be for examplemeasured through the release of cytokines, expression changes of cellsurface markers or other physiological parameters as proliferation. Thesubstance or immunomodulator is preferably an oligonucleotide.

The term “oligonucleotide” refers to a nucleic acid polymer, typicallywith from 8 to 120 bases, preferably of about 12 to about 30nucleotides. Preferably, said oligonucleotide represents a DNAoligonucleotide, which should be interpreted as being equal to anoligodeoxyribonucleotide or an oligodeoxyribonucleic acid (ODN).

The phrase “tertiary structural type” refers to different spatialorganizations that an oligonucleotide may adopt. In relation to thepresent invention, the following described types of tertiary structureare relevant: telomeric G-quadruplex tetramer type; fragile XG-quadruplex dimer type; telomeric G-quadruplex dimer type form 1;telomeric G-quadruplex dimer type form 2; non-G-quadruplex dimer type;G-quadruplex basket monomer type; random type and G-quadruplex chairmonomer type. These structural types are further described and definedbelow.

The phrase “adopts, in phosphate-buffered saline solution” refers tothat the tertiary structural type of the oligonucleotides of theinvention adopts a particular tertiary structure type that is measurablein a phosphate-buffered saline solution. A phosphate-buffered salinesolution refers here to a buffer solution system relevant or similar tophysiological conditions such as a phosphate-buffered saline solution(PBS) comprising 10 mM phosphate buffer (pH 7.4) with 140 mM NaCl and 27mM KCl. Other phosphate-buffered saline solutions or aqueous buffersolutions with similar physiological properties may also be used.

The experiments related to the present invention are typically carriedout at room temperature. However, they can also be carried out at 37° C.

The testing of the capability for oligonucleotides of the invention tomodulate the immune system in mammalian subjects, may be carried out asdescribed below in Example 2.

The invention relates to the surprising concept that if a certainoligonucleotide forms a certain tertiary structure, at least to acertain percentage in a composition, it is capable of modulating theimmune system, such as increasing levels of cytokines. Therefore, thereis provided methods for identification of such oligonucleotides. Inorder to determine the tertiary structure, samples were prepared andanalyzed by circular dichroism (CD) measurement. The results werecompared to the results of CD measurements of a number of referenceoligonucleotides that are capable of, and established to form particulartertiary structures. The sequences of these reference oligonucleotidesare disclosed in Table 1.

TABLE 1 Tertiary structure references for CD spectra analysis. SEQ IDStructure Ref. IDX- NO Sequence 5′-3′ type number No 1 TGGGGTtelomeric G- #1 0400 quadruplex tetramer 2 GCGGTTTGCGG fragile X G- #20405 quadruplex dimer 3 GGGTTTTGGG telomeric G- #3 0415 quadruplexdimer form 1 4 GGGGTTTTGGGG telomeric G- #4 0420 quadruplex dimer form 25 GCATGCT non-G- #5 0430 quadruplex dimer 6 GGTTTTGGTTTTGG G-quadruplex#6 0435 TTTTGG basket monomer 7 T*C*A*CGACCGTC random #7 9011 AAAC*T*C*C8 GGTTGGTGTGGTTGG G-quadruplex #8 0410 chair  monomer *=phosphorothioate modification

The properties set out in Table 1, and adhered to throughout thisspecification, are to be understood as:

Telomeric G-Quadruplex Tetramer Type

Oligonucleotide TGGGGT is derived from the O. nova telomeric sequence.Previous work using both X-ray crystallographic (XRC) and nuclearmagnetic resonance (NMR) has shown that it forms a tetrameric structure(see e.g. Phillips, K., et al. (1997) J. Mol. Biol., 273, 171-182;Aboul-ela, F. et al. (1994) J. Mol. Biol., 243, 458-471; Aboul-ela, F.et al. (1992) Nature, 360, 280-282.). Thus, the oligonucleotide TGGGGT(SEQ ID NO 1—reference #1) serves as reference oligonucleotide for atelomeric G-quadruplex tetramer type and its CD spectrum is depicted inFIG. 1A.

Fragile X G-Quadruplex Dimer Type

Oligonucleotide GCGGTTTGCGG represents the fragile X gene repeatsequence and has been shown to form a specific dimeric structure (seeKettani, A. et al. (1995) J. Mol. Biol., 254, 638-65). Thus, theoligonucleotide GCGGTTTGCGG (SEQ ID NO 2—reference #2) serves asreference oligonucleotide for a Fragile X G-quadruplex dimer type andits CD spectrum is depicted in FIG. 1B.

Telomeric G-Quadruplex Dimer Type Form 1

Oligonucleotide GGGTTTTGGG is derived from the O. nova telomericsequence. NMR studies showed that it forms a specific dimericG-quadruplex structure (see e.g. Scaria, P. V. et al. (1992) Proc. NatlAcad. Sci. USA, 89, 10336-10340; Keniry, M. A. et al. (1995) Eur. J.Biochem., 233, 631-643; Hud, N. V. et al. (1996) Biochemistry, 35,15383-15390). Thus, the oligonucleotide GGGTTTTGGG (SEQ ID NO3—reference #3) serves as reference oligonucleotide for a telomericG-quadruplex dimer type form 1 and its CD spectrum is depicted in FIG.1C.

Telomeric G-Quadruplex Dimer Type Form 2

Oligonucleotide GGGGTTTTGGGG is derived from the O. nova telomericsequence. Both XRC and NMR studies showed that it forms a specificG-dimeric non-GC structure (see e.g. Schultze, P. et al. (1999) NucleicAcids Res., 27, 3018-3028; Kang, C. et al. (1992) Nature, 356, 126-131;Smith, F. W. and Feigon, J. (1993) Biochemistry, 32, 8682-8692; Haider,S. et al. (2002) Mol. Biol., 320, 189-200). Thus, the oligonucleotideGGGGTTTTGGGG (SEQ ID NO 4—reference #4) serves as referenceoligonucleotide for a telomeric G-quadruplex dimer type form 2 and itsCD spectrum is depicted in FIG. 1D.

Non-G-Quadruplex Dimer Type

Oligonucleotide GCATGCT forms a specific quadruplex dimeric structurethat does not involve G-quadruplex formation (see Leonard, G. A. et al.(1995) Structure, 3, 335-340). Thus, the oligonucleotide GCATGCT (SEQ IDNO 5—reference #5) serves as reference oligonucleotide for anon-G-quadruplex dimer type and its CD spectrum is depicted in FIG. 1E.

G-Quadruplex Basket Monomer Type

Oligonucleotide GGTTTTGGTTTTGGTTTTGG forms a specific monomericG-quadruplex structure that was shown using NMR analysis (see Marathias,V. M. and Bolton, P. H. (1999) Biochemistry, 38, 4355-4364). Thus, theoligonucleotide GGTTTTGGTTTTGGTTTTGG (SEQ ID NO 6—reference #6) servesas reference oligonucleotide for a G-quadruplex basket monomer type andits CD spectrum is depicted in FIG. 1F.

Random Type

Oligonucleotide T*C*A*CGACCGTCAAAC*T*C*C designed by the inventors andis characterised by CD spectroscopy. It shows CD spectra characteristicfor DNA that has random coil structure but doesn't form any particularspecific tertiary structure. The oligonucleotideT*C*A*CGACCGTCAAAC*T*C*C (SEQ ID NO 7—reference #7) thus serves asreference oligonucleotide for a random type and its CD spectrum isdepicted in FIG. 1G.

G-Quadruplex Chair Monomer Type

Oligonucleotide GGTTGGTGTGGTTGG is known as the thrombin-bindingaptamer. It was created by an in vitro selection approach. It forms amonomeric G-quadruplex structure that was shown by both NMR and crystalstructures (see Padmanabhan, K. and Tulinsky, A. (1996) ActaCrystallogr. D, 52, 272-282; Kelly, J. A. et al. (1996) J. Mol. Biol.,256, 417-422; Macaya, R. F. et al. (1993) Proc. Natl Acad. Sci. USA, 90,3745-3749; Padmanabhan, K. et al. (1993) J. Biol. Chem., 268,17651-17654). The oligonucleotide GGTTGGTGTGGTTGG (SEQ ID NO 8—reference#8) thus serves as reference oligonucleotide for a G-quadruplex chairmonomer type and its CD spectrum is depicted in FIG. 1H.

In a first aspect of the invention, there is provided a method foridentifying an oligonucleotide capable of modulating the immune systemin a mammalian subject comprising analyzing which tertiary structuraltype said oligonucleotide adopts, in phosphate-buffered saline solution.In said method, said capability of modulating the immune system can beanalyzed by testing whether said oligonucleotide modulates the immunesystem in a mammalian subject. Said testing may be carried out in vitro.

In one embodiment of this aspect, there is provided a method foridentifying an oligonucleotide capable of modulating the immune systemin a mammalian subject comprising analyzing which tertiary structuraltype said oligonucleotide adopts, in phosphate-buffered saline solution,wherein said modulation of the immune system represents an increase oflevels of cytokines, such as interferon-α, interferon-β, interferon-γ,IL-6 and/or IL-10.

The modulation of the immune system may represent an increase orbalancing the levels of cytokines. Preferably, said modulation of theimmune system represents an increase of the levels of cytokines.

In another embodiment of this aspect, said oligonucleotide adopts, inphosphate-buffered saline solution, a tertiary structure of at least 45%of non-G-quadruplex dimer type. Preferably, said oligonucleotide adopts,in phosphate-buffered saline solution, at least 55%, more preferably80%, even more preferably 90% of non-G-quadruplex dimer type. Saidoligonucleotide are capable of modulation of the immune system byincreasing levels of cytokines, in particular IL-6 and/or IL-10.

In another embodiment of this aspect, there is provided a method,wherein the amount of tertiary structural non-G-quadruplex dimer type isestimated by quantifying against the tertiary structural type of areference oligonucleotide that adopts, in phosphate-buffered salinesolution, non-G-quadruplex dimer type. Said reference oligonucleotidemay represent an oligonucleotide of SEQ ID NO 5. The quantifyingestimate is carried out such that the amount of structuralnon-G-quadruplex dimer type of the reference oligonucleotide is set at100 percent.

In another embodiment of this aspect, there is provided anoligonucleotide, identifiable by the methods of said methods. Saidoligonucleotide, preferably has at least one nucleotide that has aphosphate backbone modification. Said phosphate backbone modificationpreferably is a phosphorothioate or phosphorodithioate modification.Said oligonucleotide typically comprises of about 8 to about 120nucleotides, preferably of about 12 to about 30 nucleotides.

In another embodiment of this aspect, said oligonucleotide adopts, inphosphate-buffered saline solution, a tertiary structure of at least 40%of telomeric G-quadruplex tetramer type. Preferably, saidoligonucleotide adopts, in phosphate-buffered saline solution, at least60%, preferably 80%, more preferably 90% of telomeric G-quadruplextetramer type. Said oligonucleotide is capable of modulation of theimmune system by increasing levels of cytokines, in particularinterferons, such as interferon-α, interferon-β and/or interferon-γ. Themodulation of the immune system may represent an increase or balancingthe levels of said cytokines. Preferably, said modulation of the immunesystem represents an increase of the levels of said cytokines. Theamount of tertiary structural telomeric G-quadruplex tetramer type isestimated by quantifying against the tertiary structural type of areference oligonucleotide that adopts, in phosphate-buffered salinesolution, a telomeric G-quadruplex tetramer type. Said referenceoligonucleotide may represent an oligonucleotide of SEQ ID NO 1. Thequantifying estimate is carried out such that the amount of structuraltelomeric G-quadruplex tetramer type of the reference oligonucleotide isset at 100 percent.

In another embodiment of this aspect, there is provided anoligonucleotide, identifiable by the methods of the invention. Saidoligonucleotide, preferably has at least one nucleotide that has aphosphate backbone modification. Said phosphate backbone modificationpreferably is a phosphorothioate or phosphorodithioate modification.Said oligonucleotide typically comprises of about 8 to about 120nucleotides, preferably of about 12 to about 30 nucleotides.

In another aspect of the invention, there is provided a method foridentifying an oligonucleotide capable of modulating the immune systemin a mammalian subject comprising analyzing which tertiary structuraltype said oligonucleotide adopts, in phosphate-buffered saline solution,wherein said modulation of the immune system represents an increase ordecrease of levels of cell surface markers.

In another aspect of the invention, there is provided a method foridentifying an oligonucleotide capable of modulating the immune systemin a mammalian subject comprising analyzing which tertiary structuraltype said oligonucleotide adopts, in phosphate-buffered saline solutionwherein said modulation of the immune system represents a change in theproperties or behaviour of polymorphonuclear cells.

In another aspect of the invention, there is provided an oligonucleotideselected from the group consisting of SEQ ID NOs 60, 62, 67, 68, 70, 72,74-77 and 79-80.

In another aspect of the invention, there is provided an oligonucleotideselected from the group consisting of SEQ ID NOs 76, 77 and 80.

In one embodiment of this aspect, there is provided said oligonucleotidefor use in therapy.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in treating a disease where increasing orbalancing the levels of cytokines are beneficial for said treatment.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in treating a disease where increasing thelevels of cytokines are beneficial for said treatment.

In another aspect of the invention, there is provided an oligonucleotideselected from the group consisting of SEQ ID NOs 13-17, 19-22, 24-30,and 33-35.

In another aspect of the invention, there is provided an oligonucleotideselected from the group consisting of SEQ ID NOs 16, 28 and 33.

In one embodiment of this aspect, there is provided said oligonucleotidefor use in therapy.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in treating a disease where increasing orbalancing the levels of cytokines are beneficial for said treatment.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in treating a disease where increasing thelevels of cytokines are beneficial for said treatment.

The methods of the invention have been tested and a number of noveloligonucleotides have been shown to being capable of modulating theimmune system. The invention, therefore, makes available specific noveloligonucleotides. In one aspect of the invention there is providedoligonucleotides with sequences according to any one of sequences inTable 2.

In another aspect of the invention there is provided isolatedoligonucleotides with sequences according to any one of SEQ ID NOs13-17, 19-22, 24-30 and 33-35. These oligonucleotides have beenidentified to adopt, in phosphate-buffered saline solution, a tertiarystructure composed of at least 45% of non-G-quadruplex dimer type.Further, these oligonucleotides have also been shown to be or of beingcapable of increasing levels of cytokines, such as IL-6 and/or IL-10.These particular oligonucleotides are presented in Table 2, whichcorrelates the SEQ ID NOs with the nucleotide sequences andcorresponding internal reference codes (“IDX-No”).

TABLE 2 Active oligonucleotides with at least 45% of non-G-quadruplex dimer type. SEQ ID IDX- NO Sequence 5′-3′ No  9TCGTCGTTCTGCCATCGTCGTT 0470 10 T*T*G*TTGTTCTGCCATCGTC*G*T*T 0475 11T*G*C*TGCTTCTGCCATGCTG*C*T*T 0480 13 T*C*G*TTCGTCTTGTTCGTTTGTTCG*T*G*G9013 14 T*C*G*TTCGTCTTTTCGTTTTCGTCGG*C*G*C 9014 15T*C*C*GCGTTCGTTGTTCGTCG*C*G*G 9017 16 C*G*G*CGCGCCGTTCGTCGA*T*G*G 902817 C*G*G*CGCCGTTCGTCGA*T*G*G 9029 18 T*C*G*TCTGCTTGTTCGTCTTGTTC*G*T*C9069 19 T*C*G*TTCGTCTGCTTGTTCGTCTTGTTC*G*T*C 9070 20T*C*G*TTCGTCTTGTTCGTCGTC*T*G*C 9072 21 T*C*G*TTCGTCTTGTTCGTC*T*G*C 907322 T*T*T*TCGTCTGCTTTCGTTTCG*T*T*T 9091 23T*G*C*C*A*T*T*C*G*T*C*G*T*T*C*T*C*G* 9100 T*C*G*T*T 24T*C*G*TCGTTCTGCCATCGT*C*G*T 9138 25 T*C*G*TCGTTCTCGTC*G*T*T 9139 26T*C*G*TTCTGCTGAT*C*G*T 9140 27 T*C*G*TCGTTCTGTCGTC*G*T*T 9141 28T*C*G*TCGTTCGTCGTC*G*T*T 9142 29 T*C*G*TCGTTGCTCGTC*G*T*T 9143 30T*C*G*TCGTTCTCGT*C*G*T 9144 33 T*C*G*TCGTTCGTCGTTCGT*C*G*T 9147 34T*T*C*TCGTTCTGCCATCGT*G*A*T 9148 35 T*C*G*TTCCGCCGAT*C*G*T 9149 44T*C*G*TCGTTCTGCCATCGTC*G*T*T 9022 45 T*C*G*TTCGTCTTGTTCGTCTTGTTC*G*T*C9012 47 T*C*G*TTCGTCTGCTTGTTC*G*T*C 9071 48T*C*C*GCGTTCGGCCTCCTGGCG*C*G*G 0001 49 T*G*C*CATTCGTCGTTCTCGTC*G*T*T9024 50 T*C*G*TCGTTCGGCCGATCG*T*C*C 9038 51 T*C*G*TTCGTCTTTCGTC*T*G*C9074 53 T*T*T*CGTCTGCTTTCGTTTCG*T*T*T 9092 54 T*C*G*TCTGCTTTCGTC*T*G*C9095 56 T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T* 0910 TG**C*G*T*T 57T*C*G*TCGTTTTGTCGTTTTGTC*G*T*T 0912 *= phosphorothioate modification

A number of oligonucleotides that do not modulate the immune system, inparticular, they do not increase levels of cytokines in the presentedassays are set out in Table 3. The tertiary structural types of theseoligonucleotides were identified and it was shown that they had adifferent tertiary structural composition than the activeoligonucleotides set out in Table 2. For these reasons, theoligonucleotides in Table 3 are designated as oligonucleotides with lessthan 45% non-G-quadruplex dimer type contribution.

TABLE 3 Oligonucleotides with less than 45% non-G-quadruplex dimer type contribution. SEQ  ID IDX- NO Sequence 5′-3′ No 7 T*C*A*CGACCGTCAAAC*T*C*C 9011 59 G*G*G*GTGCTCTGC*G*G*G 0465 31A*A*C*GACGATGGCAGAACGA*C*G*A 9153 32 A*A*G*CAGCATGGCAGAAGCA*G*C*A 914643 G*C*C*TACTAAGTAATGACTGTC*A*T*G 0495 *= phosphorothioate modification

In another aspect of the invention there is provided isolatedoligonucleotides with sequences according to any one of sequences inTable 4, particularly isolated oligonucleotides with sequences accordingto any one SEQ ID NOs 60, 62, 67, 68, 70, 72, 74-77 and 79-80. Theseoligonucleotides have been identified to adopt, in phosphate-bufferedsaline solution, a tertiary structure composed of at least 40% telomericG-quadruplex tetramer type. Further, these oligonucleotides have alsobeen shown to being capable of increasing levels of interferons, such asinterferon-α, interferon-β and/or interferon-γ. These particularoligonucleotides are presented in Table 4, which correlates the SEQ IDNOs with the nucleotide sequences and corresponding internal referencecodes (“IDX-No”).

TABLE 4 Active oligonucleotides with at least 40%of telomeric G-quadruplex tetramer type. SEQ ID IDX- NO Sequence 5′-3′No 60 T*C*T*GTCGTGTCCTTCTTT*G*G*C 9008 62 T*C*G*TCGTCTGAAGCCGC*G*G*C9019 67 T*T*C*GTCGATGGCCG*G*C*C 9027 68 G*G*G*GTCGTCTGCTATCGATG*G*G*G9039 70 G*G*T*CGTCTGCGACGATCGTCG*G*G*G 9041 72 G*G*G*GTCGTCTGCT*G*G*G9047 74 G*G*G*GTCGTCTGCTC*G*G*G 9049 75 G*G*G*GTCGTCTGCCA*G*G*G 9050 76G*A*T*CGTCCGGGTCCCGG*G*G*G 9055 77 G*A*T*CGTCCGCGG*G*G*G 9057 78T*C*G*T*C*T*G*C*C*A*T*G*G*C*G* 9067 G*C*C*G*C*C 79T*C*G*TCTGCCATGGCGCGC*C*G*G 9068 80 G*A*T*CGTCCG*T*G*T 9133 81G*G*G*GTCGTCTGC*G*G*G 9054 82 GGGGTCGTCTGCGGG 0440 83 G*A*T*CGTCCG*G*G*G9059 84 G*G*G*GTCGCAGCT*G*G*G 9004 85 T*C*G*TCCATGGTCAGGGTCCCGG*G*G*G9005 89 G*G*G*TCGTCTG*C*G*G 9053 90 G*A*T*CGTCCGTCGG*G*G*G 9058 *=phosphorothioate modification

A number of oligonucleotides that do not modulate the immune system, inparticular, they do not increase levels of interferons in the presentedassays, are set out in Table 5. The tertiary structural types of theseoligonucleotides were identified and it was shown that they had adifferent tertiary structural composition than the activeoligonucleotides set out in Table 4. For these reasons, theoligonucleotides in Table 5 are designated as oligonucleotides with lessthan 40% contribution of telomeric G-quadruplex tetramer type.

TABLE 5 Oligonucleotides with less than 40% contribution of telomeric G-quadruplex tetramer type. SEQ ID IDX- NOSequence 5′-3′ No 91 G*G*G*G*T*C*G*T*C*T*G*C*G*G*G 0445 31A*A*C*GACGATGGCAGAACGA*C*G*A 9153 32 A*A*G*CAGCATGGCAGAAGCA*G*C*A 914659 G*G*G*GTGCTCTGC*G*G*G 0465 94 G*A*T*GCTCTG*G*G*G 9134  7T*C*A*CGACCGTCAAAC*T*C*C 9011 43 G*C*C*TACTAAGTAATGACTGTC*A*T*G 0495 *=phosphorothioate modification

In another aspect of the invention there is provided an isolatedoligonucleotide which adopts, in phosphate-buffered saline solution, atertiary structure composed of at least 45% of non-G-quadruplex dimertype, said oligonucleotide being capable of balancing or increasinglevels of cytokines in a mammalian subject upon administration to saidsubject. Preferably, said non-G-quadruplex dimer type is present in atleast 55%, more preferably 80%, even more preferably 90% asnon-G-quadruplex dimer type. Preferably, said oligonucleotide is capableof increasing levels of cytokines in a mammalian subject.

In one embodiment of this aspect, said oligonucleotide comprises atleast one nucleotide that has a phosphate backbone modification.Preferably, said phosphate backbone modification is a phosphorothioateor phosphorodithioate modification.

In another embodiment of this aspect, said oligonucleotide comprises ofabout 8 to about 120 nucleotides, preferably of about 12 to about 30nucleotides.

In another embodiment of this aspect, said cytokine represents IL-6and/or IL-10.

In another embodiment of this aspect, said oligonucleotide is selectedfrom the group consisting of SEQ ID NOs 13-17, 19-22, 24-30 and 33-35.

In another embodiment of this aspect, said oligonucleotide is selectedfrom the group consisting of SEQ ID NOs 13-17 and 19-22.

In another embodiment of this aspect, said oligonucleotide is selectedfrom the group consisting of SEQ ID NOs 16, 28 and 33.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in therapy.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in treating a disease where balancing orincreasing the levels of cytokines are beneficial for said treatment.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in treating a disease where increasing thelevels of cytokines are beneficial for said treatment.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in the treatment of diseases selected frominflammatory and/or autoimmune diseases.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in the treatment of diseases selected frominflammatory bowel disease, meningitis, allergy, atopic eczema, asthmaand COPD.

In another embodiment of this aspect, there is provided use of saidoligonucleotide, in the preparation of a medicament useful in thetreatment where increasing or balancing the levels of cytokines arebeneficial for said treatment.

In another embodiment of this aspect, there is provided use of saidoligonucleotide, in the preparation of a medicament useful in thetreatment of inflammatory and/or autoimmune diseases.

In another embodiment of this aspect, there is provided use of saidoligonucleotide, in the preparation of a medicament useful in thetreatment of a disease selected from inflammatory bowel disease,meningitis, allergy, atopic eczema, asthma and COPD.

In another embodiment of this aspect, there is provided use of saidoligonucleotide, which use further comprises one or more additionalagents effective in treating inflammatory and/or autoimmune diseases.

In another embodiment of this aspect, there is provided a method oftreating a disease wherein increasing or balancing the levels ofcytokines are beneficial for said treatment, comprising administering toa subject in need thereof, of said oligonucleotide.

In another embodiment of this aspect, there is provided a method oftreating a disease wherein increasing the levels of cytokines arebeneficial for said treatment, comprising administering to a subject inneed thereof, of said oligonucleotide.

In another embodiment of this aspect, there is provided a method oftreating inflammatory and/or autoimmune diseases, comprisingadministering to a subject in need thereof, of said oligonucleotide.

In another embodiment of this aspect, there is provided a method oftreating a disease selected from inflammatory bowel disease, meningitis,allergy, atopic eczema, asthma and COPD, comprising administering to asubject in need thereof, of said oligonucleotide.

In another embodiment of this aspect, there is provided a method oftreating said disease or diseases, which further comprises one or moreadditional agents effective in treating inflammatory and/or autoimmunediseases.

The term allergy in the present invention describes an inappropriate andexcessive immunological reaction triggered by an allergen. Examples ofallergens include, but are not limited to, pollen, animal dander, dustmites, food, insect stings, microorganisms, chemicals and medications.

In yet a further aspect of the invention there is provided an isolatedoligonucleotide which adopts, in phosphate-buffered saline solution, atertiary structure composed of at least 40% of telomeric G-quadruplextetramer type, said oligonucleotide being capable of balancing orincreasing levels of interferons in a mammalian subject uponadministration to said subject. Preferably, said tertiary type ispresent in at least 60%, more preferably 80%, even more preferably 90%of telomeric G-quadruplex tetramer type. Preferably, saidoligonucleotide is capable of increasing levels of interferons.

In one embodiment of this aspect, said oligonucleotide comprises atleast one nucleotide that has a phosphate backbone modification.Preferably, said phosphate backbone modification is a phosphorothioateor phosphorodithioate modification.

In another embodiment of this aspect, said oligonucleotide comprises ofabout 8 to about 120 nucleotides, preferably of about 12 to about 30nucleotides.

In another embodiment of this aspect, said interferon representsinterferon-α, interferon-β and/or interferon-γ.

In another embodiment of this aspect, said oligonucleotide is selectedfrom the group consisting of SEQ ID NOs 60, 62, 67, 68, 70, 72, 74-77and 79-80.

In another embodiment of this aspect, said oligonucleotide is selectedfrom the group consisting of SEQ ID NOs 76, 77 and 80.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in therapy.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in treating a disease where balancing orincreasing the levels of interferons are beneficial for said treatment.Typically, said oligonucleotide for use in treating a disease is capableof where increasing the levels of interferons.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in the treatment of diseases selected frominfectious diseases, inflammatory diseases, neurodegenerative diseasesand cancer.

In another embodiment of this aspect, there is provided saidoligonucleotide for use in the treatment of diseases selected frominflammatory bowel disease, hairy cell leukemia, haematologicalmalignancy, multiple sclerosis, hepatitis B, hepatitis C, chronichepatitis, cirrhosis, chronic granulomatosis disease and severemalignant osteopetrosis.

In another embodiment of this aspect, there is provided use of saidoligonucleotide, in the preparation of a medicament useful in thetreatment where increasing or balancing the levels of interferons arebeneficial for said treatment. Preferably, said oligonucleotide iscapable of increasing the levels of interferons.

In another embodiment of this aspect, there is provided use of saidoligonucleotide, in the preparation of a medicament useful in thetreatment of infectious diseases, inflammatory diseases,neurodegenerative diseases or cancer.

In another embodiment of this aspect, there is provided use of saidoligonucleotide, in the preparation of a medicament useful in thetreatment of a disease selected from inflammatory bowel disease, hairycell leukemia, haematological malignancy, multiple sclerosis, hepatitisB, hepatitis C, chronic hepatitis, cirrhosis, chronic granulomatosisdisease and severe malignant osteopetrosis.

In another embodiment of this aspect, there is provided use of saidoligonucleotide, which use further comprises one or more additionalagents effective in treating infectious diseases, inflammatory diseases,neurodegenerative diseases or cancer.

In another embodiment of this aspect, there is provided a method oftreating a disease wherein increasing or balancing the levels ofinterferons are beneficial for said treatment, comprising administeringto a subject in need thereof, of said oligonucleotide. Preferably, saidoligonucleotide is capable of increasing the levels of interferons.

In another embodiment of this aspect, there is provided a method oftreating infectious diseases, inflammatory diseases, neurodegenerativediseases or cancer, comprising administering to a subject in needthereof, of said oligonucleotide.

In another embodiment of this aspect, there is provided a method oftreating a disease selected from inflammatory bowel disease, hairy cellleukemia, haematological malignancy, multiple sclerosis, hepatitis B,hepatitis C, chronic hepatitis, cirrhosis, chronic granulomatosisdisease and severe malignant osteopetrosis, comprising administering toa subject in need thereof, of said oligonucleotide.

In another embodiment of this aspect, there is provided a method oftreating said disease or diseases, in combination with one or moreadditional agents effective in treating infectious diseases,inflammatory diseases, neurodegenerative diseases or cancer.

In another aspect of the invention, there is provided a pharmaceuticalcomposition comprising an oligonucleotide of the invention as well asfurther oligonucleotides identifiable by the methods of the invention,together with pharmaceutically acceptable adjuvants, diluents orcarriers.

According to an embodiment, said oligonucleotide is administered in anamount of about 1 to about 2000 micro g per kg body weight, preferablyabout 1 to about 1000 micro g per kg body weight. Most preferably theoligonucleotide is administered in an amount of about 1 to 500 micro gper kg body weight.

According to another embodiment, said oligonucleotide is administered ina sufficient amount to induce immunomodulation.

In a method according to the invention, the route of administration ischosen from mucosal, subcutaneous, intramuscular, intravenous andintraperitoneal administration. According to an embodiment of themethod, the mucosal administration is selected from nasal, oral,gastric, ocular, rectal, colonic, urogenital and vaginal administration.

Nasal administration constitutes one embodiment of the method accordingto the invention. There are several methods and devices available fornasal administration; single or multi-dosing of both liquid and powderformulations, with either topical or systemic action. Using appropriatedevices or administration techniques, it is possible to target theolfactory bulb region for delivery to the CNS. The present invention isnot limited to particular methods or devices for administering theoligonucleotides to the nasal mucous membrane. The initial animalstudies have shown that simple instillation by pipette workssatisfactorily, although for human use, devices for reliable single ormulti dose of administration would be preferred.

According to another embodiment of the invention, the oligonucleotidesare administered to the mucous membrane of the colon through rectalinstillation, e.g. in the form of an aqueous enema comprising theoligonucleotides suspended in water or a suitable buffer.

According to another embodiment of the invention, the oligonucleotidesare administered to the mucous membrane of the lungs or the airwaysthrough inhalation of an aerosol, comprising the oligonucleotidessuspended in a suitable buffer, or by performing a lavage, alsocomprising the oligonucleotides suspended in water or a suitable buffer.

According to yet another embodiment of the invention, theoligonucleotides are administered to the mucous membrane of theurogenital tract, such as the urethra, the vagina etc throughapplication of a solution, a buffer, a gel, salve, paste or the like,comprising the oligonucleotides suspended in water or in a suitablevehicle.

A particular embodiment involves the use of an oligonucleotide accordingto the invention for use in conjunction with the administration of ananti cell surface marker antibody such as Rituximab. There areindications that oligonucleotides according to the invention can inducethe expression of cells surface markers, such as on immune cells such asCD20 on B cells, and thereby enhance the rate of apoptosis in humanB-cells mediated by monoclonal antibodies directed against CD20 (such asrituximab). The inventors thus make available a combination therapyinvolving the use of oligonucleotide compounds together with an anticell surface marker antibody.

A particular embodiment involves the use of an oligonucleotide accordingto the invention for use in conjunction with the administration ofglucocorticosteroids (GCS). There are indications that theoligonucleotides according to the invention can enhance GCS action. Theinventors thus make available a combination therapy involving the use ofoligonucleotide compounds together with a GCS. This is contemplated tobe able to reduce GCS consumption, and thereby reduce the cost,side-effects and risks associated with the said GCS therapy.Consequently, in this embodiment, said compound is administered togetherwith a GCS.

A skilled person is well aware of the fact that there are severalapproaches to the treatment of inflammatory and/or autoimmune diseases.Naturally new approaches are constantly being developed, and it isconceived that the oligonucleotides, their use and methods of treatmentaccording to the present invention, will find utility also incombination with future treatments. The inventors presently believe thatthe inventive oligonucleotides, their use and methods of treatment wouldbe useful as a stand-alone therapy for inflammatory and/or autoimmunediseases. It cannot however be excluded that the inventiveoligonucleotides will have utility in combination with existing orfuture treatment therapies for these diseases.

The oligonucleotide is administered in a therapeutically effectiveamount. The definition of a “therapeutically effective dose” or“therapeutically effective amount” is dependent on the disease andtreatment setting, a “therapeutically effective amount” being a dosewhich alone or in combination with other treatments results in ameasurable improvement of the patient's condition. A skilled person candetermine a therapeutically effective amount either empirically, orbased on laboratory experiments, performed without undue burden. Thetreating physician can also determine a suitable amount, based onhis/her experience and considering the nature and severity of thedisease, as well as the patient's condition.

Another embodiment is the administration of the oligonucleotide in twoor three or more separate amounts, separated in time by about 12 hours,about 24 hours, about 48 hours, about one week and about 4 weeks.Another embodiment is the administration of the oligonucleotide prior,in parallel or after the administration of a combination therapy.

The embodiments of the invention have many advantages. So far, theadministration of an oligonucleotide in the amounts defined by theinventors has not elicited any noticeable side-effects. Further, themucosal administration is easy, fast, and painless, and surprisinglyresults in a systemic effect. It is held that this effect, either alone,or in combination with existing and future treatments, offers apromising approach to fight the diseases of interest.

In another aspect of the invention, the oligonucleotides of theinvention are useful for steroid re-sensitization.

In another aspect of the invention, the oligonucleotides of theinvention are useful to influence the properties and behaviour ofpolymorphonuclear cells, in particular the recruitment and/or migrationof polymorphonuclear cells to a site of inflammation, and that theythrough this mechanism have utility in the prevention, treatment and/oralleviation of various diseases such as ischemia.

SEQ ID NOs 12, 36-42, 46, 52, 55, 58, 61, 63-66, 69, 71, 73, 86-88 and92-93 are included in the sequence listing to provide comparativeoligonucleotide sequences.

EXAMPLES Example 1 Relative Structural Composition of CD Spectra ofOligonucleotides of the Invention

The fits of the CD spectra of eight reference oligonucleotides (Table 1and FIG. 1) overlaid on the measured CD spectra of selectedoligonucleotides of the invention are presented in FIG. 4. The tertiary,structural properties of the reference oligonucleotides were analyzed bycircular dichroism (CD) measurement and the resulting traces arepresented in FIG. 1. Prior to the CD measurements the oligonucleotidesamples were annealed by heating to 90° C. and then slow cooled in waterbath to room temperature (20° C.) over a period of 8 h. Selectedoligonucleotides were annealed by heating to 90° C. and then snap-cooledin ice-cold water bath at 4° C. and then brought to room temperature. CDmeasurements in UV range were conducted on a Jasco J-720spectropolarimeter (Jasco Corp., Tokyo, Japan). For the measurement 300μl of the samples was loaded into a quartz cuvette with 0.1 cm pathlength (total volume 400 μl). The CD spectra of the oligonucleotidesamples used for the calculation were collected at 20 μM concentrationof the oligonucleotide. The CD measurements were conducted at the rateof 100 nm/min with 4 sec response time. Seven sequential spectra weretaken for each sample with the final spectrum being the average of the 7consecutive measurements. CD spectra of oligonucleotide samples weremeasured in 1×PBS buffer (10 mM phosphate buffer (pH 7.4) with 140 mMNaCl and 27 mM KCl). The blank spectrum of 1×PBS was subtracted from theCD spectra of the samples solutions.

CD spectra were collected in the range from 190 to 350 nm at 25° C. forall samples. The temperature in the sample holder was controlled andkept constant (±0.1° C.) with the help of a Peltier element (PTC-348WI). Data collection and evaluation were carried out by the softwaresupplied with the instrument. The resulting spectra were normalized tothe concentration of the oligonucleotide and zeroed at 320 nm.

For better visualization and analysis of the tertiary structure ofoligonucleotides of the invention, a custom-built computer program wasused to calculate the relative composition of the CD spectra. Theprogram was written in MatLab (Mathworks Corp.). The algorithm is basedon the assumption that the samples spectra are the linear combinationsof the references spectra (FIG. 1). The fitting of the experimental datawere performed following Levenberg-Marquart non-linear least-squaresalgorithm. Only the data collected at high tension signal below approx.600 V were considered reliable. Only the spectra measured in wavelengthwindow from 200 to 350 nm have been included in the calculations of therelative structural composition of oligonucleotide samples.

Example 2 Method for Determination of Oligonucleotide-StimulatedCytokine Production Profile of Healthy Individuals Represented byCultured Peripheral Blood Mononuclear Cells (PBMC) PBMC Isolation,Stimulation and Cultivation

Buffy coats from healthy blood donors were obtained from the KarolinskaUniversity blood bank and used for preparation of peripheral bloodmononuclear cells (PBMC). PBMC were isolated by density centrifugationon Ficoll Paque (Amersham Biosciences A B, Uppsala, Sweden). The cellswere then further washed in PBS, and the viability and the cell numberwere determined by counting the cells in Trypan blue (Sigma Aldrich,Stockholm, Sweden). Thereafter the cells were re-suspended in completecell medium consisting of RPMI 1640 (Sigma Aldrich) supplemented with10% heat inactivated fetal calf serum (FCS, Invitrogen), 2 mML-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES(Sigma Aldrich) and 5 μg/mL gentamicin (Invitrogen). The PBMC werecultured in 48-well culture tissue plates (Becton Dickinson, FranklinLakes, N.J.) at a concentration of 5×10⁶ cells/mL with 10 μM ofoligonucleotides of the invention, with medium alone as a negativecontrol in a total volume of 400 μl/well. The cells were incubated for48 h at 37° C. in a humidified cell culture incubator (ThermoScientific, Waltham, Mass.) with 5% CO₂, in air, after which the culturesupernatants were collected and frozen at −20° C. for later cytokineanalysis.

Cytometric Bead Array Measurements and Data Analysis

Culture supernatants from PBMC stimulated with oligonucleotides of theinvention were analyzed for the presence of the cytokines IFN-γ, IL-6,and IL-10 using cytometric bead array flex kit (Becton Dickinson)according to the manufacturer's instructions on a FACSArray flowcytometer (Becton Dickinson). The data were analyzed using FCAP Arraysoftware (Becton Dickinson).

ELISA and Data Analysis

Culture supernatants from PBMC stimulated with oligonucleotides of theinvention were analyzed for the presence of IFN-α using human IFN-αMulti-subtype ELISA kit (PBL, Biomedical Laboratories, NJ, USA) andIFN-β was detected with human IFN-β ELISA kit (Fujirebio INC. Tokyo,Japan) according to the manufacturer's instructions. The absorbance wasmeasured on a microplate reader (Tecan, Männedorf, Switzerland) and thedata were analyzed using Magellan software (Tecan). One set of resultsare shown in Table 6, which shows Cytometric Bead Array (CBA) mean data(IL6, IL10, IFN-γ) from four healthy individuals and ELISA mean data(IFN-α, IFN-β) from six healthy individuals on cytokines and interferonsproduction of PBMC cultured in presence of selected oligonucleotides ofthe invention.

TABLE 6 Induction of cytokines by oligonucleotides with differentcontributions of non-G-quadruplex dimer type of tertiary structure. SEQID NO IDX-no IL-6 IL-10 IFN-α IFN-β IFN-γ 7 9011 — — 1 — — 59 0465 1 — —— — 31 9153 — — — — — 32 9146 — — 1 — — 43 0495 — — — — — 44 9022 4 4 1— — 10 0475 3 3 1 — — 11 0480 2 3 — — — 45 9012 3 5 — — — 57 0912 2 3 —— — 47 9071 4 4 — — — 48 0001 4 3 5 2 — 49 9024 3 4 — — — 50 9038 5 4 5— 1 13 9013 3 4 — — — 14 9014 3 4 — — — 15 9017 4 4 3 — — 16 9028 4 4 1— 1 17 9029 4 3 2 — — 18 9069 3 4 — — — 19 9070 3 4 — — — 20 9072 3 4 —— — 21 9073 3 4 — — — 51 9074 3 5 — — — 22 9091 3 4 — — — 53 9092 3 4 —— — 54 9095 3 4 — — — 23 9100 4 3 — — — 56 0910 3 3 — — — 24 9138 5 4 21 1 25 9139 4 3 4 1 1 26 9140 3 3 2 1 1 27 9141 5 3 3 1 1 28 9142 4 3 2— 1 29 9143 4 3 3 1 1 30 9144 5 4 3 — 1 33 9147 4 3 — — 1 34 9148 4 3 3— 1 35 9149 5 3 3 — 2

The mean concentration (pg/ml) of IFN-α and IFN-β is scored as follows;

[−]<125; 125≦[1]<250; 250≦[2]<500; 500≦[3]<750; 750≦[4]<1000; and1000≦[5]

The mean concentration (pg/ml) of IL6, IL10 and IFN-γ is scored asfollows;

[−]<65; 65≦[1]<125; 125≦[2]<250; 250≦[3]<500; 500≦[4]<750; and 750≦[5]

Another set of results are shown in Table 7, which shows Cytometric BeadArray (CBA) mean data (IL6, IL10, IFN-γ) from four healthy individualsand ELISA mean data (IFN-α, IFN-β) from six healthy individuals oncytokines and interferons production of PBMC cultured in presence ofselected oligonucleotides of the invention.

TABLE 7 Induction of cytokines by oligonucleotides with differentcontributions of telomeric G-quadruplex tetramer type of tertiarystructure. SEQ ID No IDX-No IL6 IL10 IFN-α IFN-β IFN-γ 59 0465 2 1 — — —91 0445 — — — — — 31 9153 — — — — — 32 9146 — — 1 — — 43 0495 2 1 — — —94 9134 — — — — — 7 9011 — — 1 — — 81 9054 3 2 5 5 1 83 9059 5 4 5 5 180 9133 3 3 3 — — 84 9004 3 2 5 3 1 85 9005 2 2 5 5 1 60 9008 2 3 5 1 162 9019 1 2 5 2 — 67 9027 3 3 5 2 — 68 9039 2 2 4 — 1 70 9041 4 3 5 1 172 9047 2 2 5 2 1 74 9049 3 4 5 — 1 75 9050 2 3 5 2 1 76 9055 4 3 5 5 277 9057 3 3 5 5 1 78 9067 4 2 5 1 — 79 9068 1 1 5 2 — 89 9053 5 3 5 1 190 9058 5 4 5 5 1

The mean concentration (pg/ml) of IFN-α and IFN-β is scored as follows;

[−]<125; 125≦[1]<250; 250≦[2]<500; 500≦[3]<750; 750≦[4]<1000; and1000≦[5]

The mean concentration (pg/ml) of IL6, IL10 and IFN-γ is scored asfollows;

[−]<65; 65≦[1]<125; 125≦[2]<250; 250≦[3]<500; 500≦[4]<750; and 750≦[5]

Example 3 Relation Between Tertiary Structure of Inventive Compounds andtheir Biological Activity

The relative composition of tertiary structure of representativeoligonucleotides of the invention, compared their ability to inducespecific cytokines, are shown in Table 8.

TABLE 8 Non-G-quadruplex dimer type contribution linked dominantly withIL-6/IL-10 induction. % contribution of REF #5 % % SEQ (non-G-contribution contribution ID IDX- quadruplex of REF #7 of ILs IFNs NO Nodimer) (random) other REFs 6 10 α β γ 7 9011 0.0 100.0 0.0 − − − − − 319153 14.5 85.5 0.0 − − − − − 32 9146 0.0 98.7 1.3 − − − − − 59 0465 28.05.2 66.8 − − − − − 43 0495 0.0 100.0 0.0 − − − − − 44 9022 91.6 8.4 0.0‡ + − − − 10 0475 53.6 46.4 0.0 ‡ (+) − − − 11 0480 81.4 17.8 0.0 (+)(+) − − − 57 0912 72.2 27.8 0.0 (+) + − − − 47 9071 60.2 39.2 0.6 ‡ ‡ −− − 48 0001 64.0 36.0 0.0 ‡ + − − − 49 9024 83.1 16.9 0.0 + ‡ − − − 169028 74.2 24.2 1.6 ‡ ‡ − − (+) 33 9147 97.0 0.0 3.0 ‡ + − − (+) 28 914294.7 0.0 5.3 ‡ + − − (+)

The mean concentration (pg/ml) of IL6 and IL10 is scored as follows;

[−]<125; 125≦[(+)]<350; 350≦[+]<500, 500≦[‡]

The mean concentration (pg/ml) of IFN-γ is scored as follows;

[−]<60; 60≦[(+)]<100; 100≦[+]

The data in Table 8 shows that representative oligonucleotides that arepresent in at least 45% non-G-quadruplex dimer type (related to Ref #5,SEQ ID NO 5) are capable of increasing levels of cytokines IL-6 andIL-10. The data (IL-6, IL-10, IFN-α, IFN-β and IFN-γ) are obtained inaccordance with the methods set out in Example 1. The tertiary structureof SEQ ID NO 7, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 43 and SEQ ID NO59 (Tables 6 and 8) represents less than 45% non-G-quadruplex dimer typeand, thus, according to the invention, are not capable of sufficientlyincreasing levels of cytokines IL-6 and IL-10.

The relative composition of tertiary structure of further representativeoligonucleotides of the invention, compared with their ability to inducespecific cytokines, are shown in Table 9.

TABLE 9 Telomeric G-quadruplex tetramer type contribution linkeddominantly with IFN induction. % % % contribution contributioncontribution of REF of REF of REF % % #1 (telomeric #2 (fragile × #5contribution Contribution SEQ G- G- (non-G- of of ID IDX- quadruplexquadruplex quadruplex REF #7 other IL IFN NO No tetramer) dimer) dimer)(random) REFs 6 10 α β γ 7 9011 0.0 0.0 0.0 100.0 0.0 − − − − − 32 91460.0 0.0 0.0 98.7 1.3 − − − − − 43 0495 0.0 0.0 0.0 100.0 0.0 − − − − −91 0445 15.3 0.0 0.0 49.1 35.6 − − − − − 59 0465 22.2 35.4 28.0 5.2 5.0− − − − − 94 9134 25.4 41.7 6.0 22.7 4.1 − − − − − 81 9054 61.0 0.0 14.817.5 7.7 (+) (+) ‡ + (+) 83 9059 49.0 0.0 0.0 43.2 7.8 ‡ ‡ ‡ + (+) 809133 44.3 0.0 10.1 43.8 1.8 (+) + (+) − − 76 9055 90.0 0.0 0.0 10.0 0.0‡ + ‡ + + 77 9057 64.0 0.0 0.0 36.0 0.0 + (+) ‡ + (+)

The mean concentration (pg/ml) of IFN-α and IFN-β is scored as follows:

[−]<500; 500≦[(+)]<1000; 1000≦[+]<5000; 5000≦[‡]

The mean concentration (pg/ml) of IL6 and IL10 is scored as follows;

[−]<125; 125≦[(+)]<350; 350≦[+]<500; 500≦[‡]

The mean concentration (pg/ml) of IFN-γ is scored as follows;

[−]<60; 60≦[(+)]<100; 100≦[+]

The data in Table 9 shows that representative oligonucleotides that arepresent in at least 40% telomeric G-quadruplex tetramer type (related toRef #1, SEQ ID NO 1) are capable of increasing levels of interferons.The data (IL-6, IL-10, IFN-α, IFN-β and IFN-γ) are obtained inaccordance with the methods set out in Example 1. The tertiary structureof SEQ ID NO 59, SEQ ID NO 91, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 43,SEQ ID NO 94 and SEQ ID NO 7 (Tables 7, and 9) represents less than 40%telomeric G-quadruplex tetramer type and, thus, according to theinvention, are not capable of increasing levels of interferons.

The invention claimed is:
 1. An oligonucleotide selected from the groupconsisting of SEQ ID NOs: 19, 20, 34, and
 76. 2. The oligonucleotideaccording to claim 1, wherein the oligonucleotide is at most 120nucleotides.
 3. The oligonucleotide according to claim 2, wherein theoligonucleotide is at most 30 nucleotides.
 4. An oligonucleotideselected from the group consisting of: SEQ ID NO: 19, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 27, 28, and 29; SEQ ID NO: 20, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 21, 22, and 23; SEQ ID NO: 21, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 18, 19, and 20; SEQ ID NO: 22, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 21, 22, and 23; SEQ ID NO: 26, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 13, 14, and 15; SEQ ID NO: 27, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 16, 17, and 18; SEQ ID NO: 28, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 15, 16, and 17; SEQ ID NO: 29, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 15, 16, and 17; SEQ ID NO: 33, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 18, 19, and 20; SEQ ID NO: 34, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 18, 19, and 20; SEQ ID NO: 35, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 13, 14, and 15; SEQ ID NO: 60, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 18, 19, and 20; SEQ ID NO: 62, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 17, 18, and 19; SEQ ID NO: 67, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 14, 15, and 16; SEQ ID NO: 72, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 13, 14, and 15; SEQ ID NO: 74, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 14, 15, and 16; SEQ ID NO: 75, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 14, 15, and 16; SEQ ID NO: 76, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 17, 18, and 19; and SEQ ID NO: 77, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions 1, 2, 3, 12, 13, and
 14. 5. The oligonucleotide according toclaim 4, wherein the oligonucleotide is at most 120 nucleotides.
 6. Theoligonucleotide according to claim 4, wherein the oligonucleotide is atmost 30 nucleotides.
 7. The oligonucleotide of claim 4, wherein theoligonucleotide is SEQ ID NO: 19, which comprises phosphorothioate orphosphorodithioate backbone modification at positions 1, 2, 3, 27, 28,and
 29. 8. The oligonucleotide of claim 4, wherein the oligonucleotideis SEQ ID NO: 20, which comprises phosphorothioate or phosphorodithioatebackbone modification at positions 1, 2, 3, 21, 22, and
 23. 9. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:21, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 18, 19, and
 20. 10. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:22, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 21, 22, and
 23. 11. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:26, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 13, 14, and
 15. 12. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:27, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 16, 17, and
 18. 13. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:28, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 15, 16, and
 17. 14. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:29, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 15, 16, and
 17. 15. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:33, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 18, 19, and
 20. 16. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:34, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 18, 19, and
 20. 17. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:35, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 13, 14, and
 15. 18. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:60, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 18, 19, and
 20. 19. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:62, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 17, 18, and
 19. 20. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:67, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 14, 15, and
 16. 21. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:72, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 13, 14, and
 15. 22. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:74, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 14, 15, and
 16. 23. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:75, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 14, 15, and
 16. 24. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:76, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 17, 18, and
 19. 25. Theoligonucleotide of claim 4, wherein the oligonucleotide is SEQ ID NO:77, which comprises phosphorothioate or phosphorodithioate backbonemodification at positions 1, 2, 3, 12, 13, and
 14. 26. Theoligonucleotide of claim 4, wherein the backbone modification at thepositions is phosphorothioate.
 27. An oligonucleotide selected from thegroup consisting of: SEQ ID NO: 19, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 27, 28, and 29; SEQ ID NO: 20, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 21, 22, and 23; SEQ ID NO: 21, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 18, 19, and 20; SEQ ID NO: 22, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 21, 22, and 23; SEQ ID NO: 24, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 18, 19, and 20; SEQ ID NO: 25, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 14, 15, and 16; SEQ ID NO: 26, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 13, 14, and 15; SEQ ID NO: 27, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 16, 17, and 18; SEQ ID NO: 28, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 15, 16, and 17; SEQ ID NO: 29, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 15, 16, and 17; SEQ ID NO: 30, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 13, 14, and 15; SEQ ID NO: 33, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 18, 19, and 20; SEQ ID NO: 34, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 18, 19, and 20; SEQ ID NO: 35, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 13, 14, and 15; SEQ ID NO: 60, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 18, 19, and 20; SEQ ID NO: 62, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 17, 18, and 19; SEQ ID NO: 67, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 14, 15, and 16; SEQ ID NO: 72, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 13, 14, and 15; SEQ ID NO: 74, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 14, 15, and 16; SEQ ID NO: 75, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 14, 15, and 16; SEQ ID NO: 76, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 17, 18, and 19; SEQ ID NO: 77, which comprises phosphorothioate orphosphorodithioate backbone modification at positions consisting of 1,2, 3, 12, 13, and 14; and SEQ ID NO: 80, which comprisesphosphorothioate or phosphorodithioate backbone modification atpositions consisting of 1, 2, 3, 9, 10, and
 11. 28. The oligonucleotideof claim 27, wherein the backbone modification at the positions isphosphorothioate.
 29. A method for treating a disease, wherein thedisease is selected from the group consisting of inflammatory boweldisease, meningitis, allergy, atopic eczema, asthma, COPD, infectiousdiseases, inflammatory diseases, neurodegenerative diseases, cancer,hairy cell leukemia, haematological malignancy, multiple sclerosis,hepatitis B, hepatitis C, chronic hepatitis, cirrhosis, chronicgranulomatosis disease, malignant osteopetrosis, comprisingadministering an effective amount of an oligonucleotide according toclaim 1, to a subject in need thereof, wherein an increase or balancingthe levels of cytokines is beneficial to said disease.
 30. A method fortreating a disease, wherein the disease is selected from the groupconsisting of inflammatory bowel disease, meningitis, allergy, atopiceczema, asthma, COPD, infectious diseases, inflammatory diseases,neurodegenerative diseases, cancer, hairy cell leukemia, haematologicalmalignancy, multiple sclerosis, hepatitis B, hepatitis C, chronichepatitis, cirrhosis, chronic granulomatosis disease, malignantosteopetrosis, comprising administering an effective amount of anoligonucleotide according to claim 4, to a subject in need thereof,wherein an increase or balancing the levels of cytokines is beneficialto said disease.
 31. A method for treating a disease, wherein thedisease is selected from the group consisting of inflammatory boweldisease, meningitis, allergy, atopic eczema, asthma, COPD, infectiousdiseases, inflammatory diseases, neurodegenerative diseases, cancer,hairy cell leukemia, haematological malignancy, multiple sclerosis,hepatitis B, hepatitis C, chronic hepatitis, cirrhosis, chronicgranulomatosis disease, malignant osteopetrosis, comprisingadministering an effective amount of an oligonucleotide according toclaim 27, to a subject in need thereof, wherein an increase or balancingthe levels of cytokines is beneficial to said disease.